Due to tumor heterogeneity in progression and frequent recurrence, the management of bladder cancer (BCa) patients is both challenging and costly. Hyaluronic acid (HA) is a glycosaminoglycan and HYAL1 is a tumor cell-derived hyaluronidase (HAase) that degrades HA into angiogenic fragments. HYAL1, HA and HA-synthase (HAS) are molecular determinants of BCa growth, invasion and angiogenesis, and are also accurate markers of BCa diagnosis. The objectives of this proposal are to examine the mechanism by which HA-HYAL1 system promotes BCa growth and progression, how HYAL1 gene expression is regulated in BCa, and do HA-HYAL1 family of markers have any prognostic potential for BCa patients. Sulfated HA compounds inhibit HYAL1 activity and inhibit BCa growth by causing cell cycle arrest and apoptosis. 4-methylumbelliferone (4-MU) inhibits HA synthesis and inhibits BCa cell growth by inducing apoptosis. 4-MU and sHA are non-toxic and orally bioavailable. To examine how HYAL1 and HA regulate BCa growth and progression, we will evaluate the effects of sHA compounds and 4-MU in vitro and in an orthotopic BCa model that metastasizes to lung. The intracellular mechanisms by which sHA and 4-MU induce cell cycle arrest and/or apoptosis and affect HA receptor-mediated signaling in BCa and normal cells will also be examined (Aim 1). Currently, it is unknown how HYAL1 expression is regulated in normal and tumor tissues. Cloning of HYAL1 promoter showed that DNA methylation and certain transcription factors are involved in HYAL1 gene regulation in BCa cells. To examine the mechanism of HYAL1 regulation, a promoter methylation and HYAL1 expression will be evaluated in a cohort of normal and BCa tissues. Interaction between a transcription factor and HYAL1 promoter will be evaluated in BCa cells and tissues by EMSA and ChiP assay and siRNA transfection. Regulation of methylation and HYAL1 promoter activity by HA-HYAL1 signaling will also be evaluated (Aim 2). In BCa, the expression of HA, HYAL1 and HA-syntheses (HAS1, 2, 3) is elevated. The efficacy of HA-HYAL1 family of markers to predict disease progression, tumor recurrence and treatment response will be evaluated in a multi-center retro-prospective study that involves tissue microarrays (Aim 3). This study should reveal how HA-HYAL1 promotes BCa growth and progression and whether it can be controlled by HA and HAase inhibitors. It will also reveal how HYAL1 expression is regulated in BCa tissues and may suggest ways to control it. The study should establish whether HA- HYAL1 family members can predict prognosis for BCa patients. PUBLIC HEALTH RELEVANCE: The study should reveal how HA-HYAL1 promotes BCa growth and progression and whether it can be controlled by HA and HAase inhibitors. It will also reveal how HYAL1 expression is regulated in BCa tissues and may suggest ways to control it. The study should establish whether HA-HYAL1 family members have a place in the clinical management of BCa patients for predicting prognosis.